Kim 1

For the immunohistochemical staining, paraffin-embedded sections (5 µm thick) were incubated with primary antibodies against mouse TLR2 (Abcam), TLR4 and cubilin (Santa Cruz), Kim-1 (R&D) and cleaved caspase 3 (Cell Signaling); or human TLR2 and TLR4 (Santa Cruz), HSP70 (Enzo Life Science) and HMGB1 (Abcam). Slides were developed using 3,3′-diaminobenzidine substrate-chromogen solution (Dako). For the immunofluorescence staining, frozen sections (12 µm thick) were incubated with antibodies against HSP60 and HSP70 (Enzo Life Science), and biglycan and HMGB1 (Abcam) followed by secondary antibodies conjugated with Alexa Fluor dyes (Invitrogen). For the immunobloting, proteins were probed with antibodies against Kim-1, podocin, HSP70 (Santa Cruz), HMGB1 (Abcam), nephrin (PROGEN), caspase 3 (Cell Signaling), β-actin and α-tubulin (Sigma-Aldrich).

Formalin-fixed tissues were dehydrated, cleared, and embedded in paraffin. The tissue blocks were sectioned and stained with hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS). Tubular injury score was assessed in 5 randomly selected fields per sample, as previously described [14 (link), 15 (link)]. For IHC, primary antibodies against neutrophil gelatinase-associated lipocalin (NGAL; Santa Cruz Biotechnology, Santa Cruz, CA, USA), kidney injury molecule-1 (KIM-1; Santa Cruz Biotechnology), 4-hydroxy-2-nonenal (4-HNE; Invitrogen, Carlsbad, CA, USA), F4/80 (Santa Cruz Biotechnology), and CD4 (Novus Biologicals, Littleton, CO, USA) antibodies were used. Mouse IgG1 isotype control antibody (R&D Systems) was used as a primary antibody for negative control. Positive areas were examined in 5 randomly selected fields at 400x magnification per sample using a computerized image analyzer (i-Solution DT software; IMT i-Solution, Coquitlam, BC, Canada), and the results were presented as percentage of the positively stained area with respect to the total area analyzed. Positive cells were examined in 10 randomly selected fields at 1000x magnification per sample.

Cortical kidney lysates with equal amounts of total protein (50 μg/ml) were fractionated by 7.5–15% SDS-polyacrylamide gels under reducing conditions and analyzed by immunoblot. The antibodies against fibronectin, collagen I (ab34710, Abcam), MR, arginase I (ab64693, Abcam), NGAL, KIM-1, TNF-α, p38 (sc-535, Santa Cruz Biotechnology), MCP-1, phosphorylated-p38 (p-p38, 9211, Cell Signaling Technology, Danvers, MA, USA), ERK1/2 (sc-292838, Santa Cruz Biotechnology), GSK3β (sc-53931, Santa Cruz Biotechnology), phosphorylated GSK 3β-Ser 9 (p-GSK3β-Ser 9, sc-11757, Santa Cruz Biotechnology), phosphorylated ERK (p-ERK 1/2, 9101, Cell Signaling Technology), JNK (sc-572, Santa Cruz Biotechnology), phosphorylated JNK (p-JNK, ab124956, Abcam) and GAPDH (sc-48166, Santa Cruz Biotechnology) were used as primary antibodies.

Kidney tissue lysates (40 μg) were run on 10% SDS-polyacrylamide gel electrophoresis, subsequently transferred to nitrocellulose membrane. Blots were blocked with 5% BSA and then exposed to the appropriate primary antibody in dilutions suggested from the commercial supplier. Primary antibody KIM-1, NGAL, TNF-α, IL-1β, IL-6, cryopyrin (NLRP3), ASC, pro-caspase-1, and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Primary antibodies were probed with horseradish peroxidase conjugated goat anti-rabbit-IgG or anti-mouse-IgG. Visualization was performed by chemiluminescence (EzWestLumi plus, ATTO, NY, USA). Capturing image was achieved by gel image system (iBright FL100, Thermo Fisher Scientific, Waltham, MA, USA).

CP, TSA, dimethyl sulfoxide, VPA, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) were obtained from Sigma-Aldrich (St. Louis, MO, USA). BMP-7, KIM-1, Bcl-2 and Bax antibodies were purchased from Santa Cruz (Santa Cruz biotechnology, Dallas, TX, USA). β-Actin antibody and secondary antibodies for goat anti-rabbit IgG HRP and rabbit anti-goat IgG HRP were purchased from Bioworld Technology (Nanjing, China). Cr and BUN assay kit were purchased from Siemens (Siemens Healthcare Diagnostics, Tarytown, NY, USA).

Whole protein extracts were separated by 10 or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes, which were incubated with primary antibodies against KIM-1 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA). The membranes were then washed in TBS/Tween 20 and incubated with secondary antibodies correspondingly. After extensive washing in TBS/Tween 20, protein bands were visualized with an ECL chemiluminescent kit (ECL-plus; Thermo Fisher Scientific, Pittsburgh, PA, USA).

Cisplatin (CP), glycyrrhizic acid ammonium salt (GA), 18β-glycyrrhetinic acid (18βGA), Dimethyl sulfoxide (DMSO), MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide) and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). BMP-7, KIM-1, Bcl-2 and Bax antibodies were purchased from Santa Cruz (CA, USA). β-actin antibody and secondary antibodies for goat anti-rabbit IgG HRP and rabbit anti-goat IgG HRP were purchased from Bioworld Technology (Nanjing, China). Creatinine (Cr) and Blood Urea Nitrogen (BUN) assay kit were purchased from Siemens (NY, USA).